*3.1.3 Introducing the extracellular electron transfer pathway*

*Current Topics in Biochemical Engineering*

**3.1 Modification of microorganisms**

catalyst development are discussed.

*3.1.1 Chemical treatment*

density (41 mW/m2

MFC performance.

*3.1.2 Biosurfactant production by gene modification*

important components for the electron transfer [53]. On the other hand, *G. sulfurreducens* has electrically conductive pili, called nanowires, which can transfer electrons to extracellular electron acceptors on the cell surface [54]*. S. oneidensis* also has an electrically conductive structure similar to the pili, but its structure is different, whereby the membrane structure containing the cytochrome protein described above was raised [54]. In any case, it has been confirmed that electrons can be delivered via such protrusions. It is expected that new developments will be made in the future, such as introducing the genes related to these mechanisms into

other species, especially model organisms, such as *E. coli* and *S. cerevisiae*.

The utilization of chemical and biotechnological techniques is important to modify the microbial cells as biocatalysts in the MFC system. Molecular biology approaches are effective tools to improve the performance of the biocatalysts for the desired system. In this section, recent topics about the approaches for microbial

Mediators and macromolecular catabolic enzymes, which are used for electron transfer and other metabolic activities, are abundant in the cytoplasm of the microbial cells used in the MFCs. However, it is not easy to transport the mediator molecules to the bacterial outer membrane so as to reach the electrode. The lipopolysaccharide (LPS) layer on the Gram-negative outer membrane is compact and nonconductive; thus, most microbial cells are nonconductive [55]. It was found that chemically perforated pores and channels on the cell membranes accelerated electron transfer, leading to an improved power output for an MFC using *P. aeruginosa* [56]. In their study, polyethyleneimine (PEI)-treated biofilm achieved a doubled power

nels on the cell membrane created by PEI treatment promoted the diffusion of the self-produced mediators (pyocyanin and pyorubin) of *P. aeruginosa*. The modified cell membrane surface also promoted the adherence of microbial cells to the electrode, which further improved the electron transfer. This method was also applied to *E. coli* [57]. Recently, it has been reported that lysozyme treatment increased 1.75-fold of the MFC performance with *K. rhizophila* P2-A-5 [58]. Thus, chemical treatment is one of the important approaches to modify the microbial cells for the improvement of the

To increase the cell permeability of biocatalysts in the MFCs, Zheng et al. proposed a new approach by inducing the biosurfactant production based on a genetic modification [59]. It is true that the efficiency of membrane permeability can be improved with a biosurfactant, which ultimately increases the transport across the membrane. In addition, overexpression of the *rhlA* gene, which is responsible for rhamnolipid (a biosurfactant) production, was also conducted [60]. The biosurfactant directly influenced the overproduction of rhamnolipids from the electrical bacteria, such as *P. aeruginosa*. The electron transport across the membrane was greatly increased as the membrane permeability increased. The power output of the

) compared to the control biofilms. The large pores and chan-

**3. Recent topics of microbial catalyst and future directions**

**58**

The sparse availability of genetic tools in manipulating electricity-generating bacteria and the multiple overlapping pathways for extracellular electron transfer make it challenging to modulate electron transfer and/or introduce other functions of interest. In response to this challenge, several studies have taken the complementary approach of engineering portions of the extracellular electron transfer pathways into the well-studied industrial microbe *E. coli* [61]. In these studies, MtrCAB of *S. oneidensis* was successfully expressed in the *E. coli* cells, and the activity of these proteins was confirmed by the metal reduction. Although the introduction of MtrCAB permits extracellular electron transfer in *E. coli*, the low electron flux and the absence of growth in these cells limit their practical application. Recently, in addition to surface-localized cytochromes, it has been further confirmed that CymA, the inner membrane component of *S. oneidensis*, significantly improved the extracellular electron transfer rate or cell viability. This recombinant *E. coli* achieved current generation in an MFC system without the addition of mediators [62, 63]. Our research group is trying to develop an excellent *E. coli* biocatalyst for the anode in an MFC system based on the combination of engineering of central metabolism and introduction of extracellular electron transfer in the presence of an HNQ mediator.
